Method for diversifying the chemical composition of proteins produced in vivo by genetically disabling the editing function of their aminoacyl tRNA synthetases

ABSTRACT

The present invention is directed to a method to diversify the chemical composition of proteins produced in vivo comprising the step of disabling, particularly by mutagenesis, the editing function of one of its aminoacyl tRNA synthetases. The present invention is also directed to nucleic acid sequences encoding such mutated aminoacyl tRNA synthetases having their editing site mutated and capable of mischarging its cognate tRNA with a noncanonical amino acid. Also described herein is an improved method for obtaining transformed cells capable of synthetizing in vivo proteins comprising at least a noncanonical amino acid and their use for the production of such proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of earlier-filed U.S.provisional application serial No. 60/285,495, filed Apr. 19, 2001,which application is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention is directed to a method to diversify thechemical composition of proteins produced in vivo, especially to methodscomprising the step of disabling, particularly by mutagenesis, theediting function of one of its aminoacyl tRNA synthetases. The presentinvention is also directed to nucleic acid sequences encoding suchmutated aminoacyl tRNA synthetases having their editing site mutated andcapable of mischarging its cognate tRNA with a noncanonical amino acid.Also described herein is an improved method for obtaining transformedcells capable of synthesizing in vivo proteins comprising at least anoncanonical amino acid and their use for the production of suchproteins.

BACKGROUND OF THE INVENTION

[0003] Aminoacyl tRNA synthetases establish the rules of the geneticcode by catalyzing the aminoacylation of transfer RNAs. The chemicalinvariance of the twenty amino acid building blocks of proteins is wellestablished. The only known extensions to this invariant set areformyl-methionine (1) and selenocysteine (2), both incorporated inresponse to punctuation signals during translation in certain organisms.Thus, although species have colonized dissimilar terrestrial habitatsthroughout geological times, this diversification has not been mirroredin the evolution of organisms to include specialized sets of aminoacids. For instance, thermophilic, mesophilic, and psychrophilicorganisms all assemble proteins by combining the same types of twentycanonical amino acids into different protein sequences. Standing as the“missing link” between alanine and valine (3), aminobutyrate (Abu, alsoknown as butyrine) can be generated by transamination from thephysiological metabolite 2-oxo-butyrate and should thus be considered asa latent metabolite (4). Its absence is therefore particularlyconspicuous in the proteins of extant organisms.

[0004] The selection of amino acids for protein synthesis is done byaminoacyl tRNA synthetases. Typically, each of twenty synthetasescatalyzes the attachment of its cognate amino acid to the 3′-end of itscognate tRNA and amino acids are, in this way, associated with specifictriplets of the genetic code (5). The active site of several of theseenzymes inherently lack the capacity to discriminate between closelysimilar amino acids at a level sufficient to explain the high accuracyof the code. For that reason, a given enzyme may misactivate closelysimilar (in size and shape) amino acids at a low frequency (0.1 to 1%)(6). To correct these errors, in many cases, a hydrolytic editingfunction, at a separate active site, has developed (7-10). One exampleof a synthetase that has editing activity is valyl-tRNA synthetase(ValRS), which misactivates the isosteric natural amino acid Thr (9), aswell as the non-natural Abu Val (11). Misactivation of these amino acidsleads to transient mischarging of tRNA followed by hydrolyticdeacylation (editing) of the mischarged amino acid from the tRNA.

[0005] The present work aimed to establish conditions of artificialselection that promoted usage of non-canonical amino acids, such as Abu,that were not retained by natural selection. Others have attempted toincorporate a non-canonical amino acid into a protein by introducing aforeign, “orthogonal” tRNA/synthetase pair that can insert the aminoacid at a specialized stop codon (12).

[0006] However, such approaches are laborious, as they requireselection, identification, cloning, and study of individual mutantstrains.

[0007] In order to facilitate the in vivo production of proteinscomprising noncanonical amino acids, it would be desirable to have arapid and generalized method allowing to genetically modify and selectcells capable of achieving the in vivo production of such proteins.

[0008] Such a desirable method will allow to enlarge the chemistry oftranslation by having a non-canonical amino acid “infiltrate” all of thecodons normally associated with one of the natural amino acids. Indeed,by assigning two amino acids (a cognate and a non-cognate) to a specificset of codons so as to provide a selective advantage to the reprogrammedcells, global changes in the amino acid compositions of all cellularproteins could be made. The present invention addresses this need.

SUMMARY OF THE INVENTION

[0009] The present invention provides a general method to diversify thechemical composition of proteins produced in vivo by a cell comprisingthe step wherein the editing function of an aminoacyl tRNA synthetase ofsaid cell has been disabled.

[0010] This rapid and general method may be used to obtain cellscomprising a mutation in the DNA sequence encoding the editon domain ofsaid disabled aminoacyl tRNA synthetase compared to the wild typeaminoacyl tRNA synthetase coding sequence.

[0011] In general, the method of the present invention includes: a)selecting a cell strain wherein the editing function of at least one ofthe cell's aminoacyl tRNA synthetases has been disabled, said disabledediting function allowing the aminoacyl tRNA synthetase to mischarge thecognate tRNA with said at least one noncanonical amino acid; b)culturing the selected strain in a culture medium comprising saidnoncanonical amino acid, or one of its precursor, under conditionsfavourable for the growth of said strain; and c) recovering from theculture medium or from the cells obtained in step b) the proteinscontaining said noncanonical amino acid.

[0012] In various embodiments, the editing function of the aminoacyltRNA synthetases has been disabled by mutagenizing the DNA sequenceencoding the editing domain of an aminoacyl tRNA synthetase in thetarget cell, said mutagenesis being carried out in the cell preferablyby homologous recombination or allele replacement vector leading to anaminoacyl tRNA synthetase variant having an amino acid mutation in itsediting domain, said mutation allowing the aminoacyl tRNA synthetasevariant to mischarge its cognate tRNA with one noncanonical amino acid.

[0013] In a particular related aspect, the present invention is directedto a method for selecting an aminoacyl tRNA synthetase variant capableof mischarging its cognate tRNA with a noncognate amino acid, preferablya noncanonical amino acid, including the steps of: a) elaborating a DNAconstruct encoding an aminoacyl tRNA synthetase variant having an aminoacid mutation in its editing domain; b) transforming a host cell withsaid DNA construct; c) assaying the ability of the recombinant aminoacyltRNA synthetase variant produced by said transformed host cell for itsability to mischarge its cognate tRNA with a noncognate amino acid,preferably a noncanonical amino acid; and d) if appropriate, selectingthe assayed aminoacyl tRNA synthetase variant if said assayed aminoacyltRNA synthetase variant is capable of mischarging its cognate tRNA(tRNA(s) associated with the assayed aminoacyl tRNA synthetase) with anoncognate amino acid, preferably with a noncanonical amino acid.

[0014] In a further aspect, the invention relates to isolated aminoacyltRNA synthetase variants capable of mischarging its cognate tRNA with anoncognate amino acid, preferably with a noncanonical amino acid,wherein the nucleic fragment encoding the editing site comprises atleast one mutation leading to an amino acid mutation, preferably anamino acid substitution, in the editing site of said aminoacyl tRNAsynthetase. Isolated nucleic acid encoding such aminoacyl tRNAsynthetase variants, vectors, such as plasmid, and cell comprising suchnucleic acid also form part of the present invention.

[0015] In another preferred embodiment, the present invention isdirected to a method for the production of proteins comprising anoncanonical amino acid including the general steps of: a) culturing, ina culture medium containing said noncanonical amino acid, or one of itsprecursors, a transformed host cell comprising an aminoacyl tRNAsynthetase allele variant capable of mischarging its cognate tRNA withsaid noncanonical amino acid; and b) recovering and, if appropriate,purifying the proteins comprising said noncanonical amino acid from theculture medium (supernatant) and/or from the cells (from cells pellet)of step a).

[0016] In a final aspect, the invention provides proteins comprising anoncanonical amino acid obtained by the above method.

[0017] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof, andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIGS. 1A to 1C. Suppression and toxicity phenotypes. Amino acidgradient plates were prepared using minimal medium (27).

[0019]FIG. 1A: A schematic showing the structures of cysteine,S-carbamoyl-cysteine, valine, threonine and a-aminobutyric acid.

[0020]FIG. 1B: Photographs showing the results of experimentsdemonstrating the cysteine-suppression phenotype. Cysteine (100 μl (0.4M)) was loaded in a central well after spreading and drying 0.5 ml of a5/1000 dilution of an overnight culture (in MS glucose medium containingthymidine (0.3 mM)) of 15456 (thyA::erm+ΔnrdD::kan+valS:T222P pTS13(bla+thyA:146GUA)). Ile-Val (0.3 mM) was added in one plate as acontrol. The dipeptide Ile-Val was purchased from Bachem AG (Bubendorf,Switzerland). Plates were incubated for 2 days at 30° C.

[0021]FIG. 1C: Photographs showing the results of growth of a straincarrying the valS:T222P allele in the presence of L-threonine or Abu.Minimal medium plates supplemented with thymidine (0.3 mM) werepretreated with amino acid solutions by streaking either with Thr (50 μl(0.2 M)) or Abu (50 μl (0.1 M)) vertically along the diameter of theplate to create an amino acid gradient. Mutant (β5456) and wild-type(β5419 (thyA::erm+AnrdD::kan+pTS13 (bla+thyA:146GUA)) strains were thenstreaked horizontally across the plates and incubated for 2 days at 37°C.

[0022]FIGS. 2A to 2C. Point mutations in the editing site and theirconsequences.

[0023]FIG. 2A: A schematic showing the positions of the five pointmutations isolated in the editing site of ValRS are shown. The IleRSediting site (CP1) (28, 29) that intersects the alternating β-strands(pentagons) and α-helices (rectangles) of the catalytic domain is shown.Alignment of residues in the editing sites of IleRS and ValRS is alsoshown, with the strictly conserved residues among all publishedsequences labeled with a colon. Abbreviations are Ec, Escherichia coli;Sc, Saccharomyces cerivisiae; Hs, Homo sapiens.

[0024]FIG. 2B: A graph showing misaminoacylation of tRNA^(Val) with Thrby the T222P mutant enzyme at pH 7.5 and 37° C. The wild-type (WT) andmutant alleles were cloned under the control of a PBAD promoter (30).The enzymes were partially purified from a laboratory strain lacking thechromosomal copy of the valS gene (AvalS::kan+). The purification andaminoacylation procedures were adapted from Hendrickson et al. (31).(Main panel) Misaminoacylation of tRNA^(Val) with Thr by the twoenzymes. (Inset) Aminoacylation of tRNA^(Val) with Val by the twoenzymes.

[0025]FIG. 2C: A schematic showing MALDI analysis demonstrating In vivoincorporation of aminobutyrate. The His-tagged protein AlaXp wasexpressed in two Δilv strains containing the wild-type valS or themutant valS:T222P allele, in MS medium containing Ile-Leu (0.3 mM),Ile-Val (0.02 mM) and Abu (0.2 mM). AlaXp was purified with Ni-NTAagarose (Qiagen GmbH, Hilden, Germany), cut out of a SDS-PAGEpreparative gel and prepared for MALDI and μ-LC-MS/MS mass analysis(32). The MALDI-MS analyses were performed in a Voyager-Elitetime-of-flight mass spectrometer with delayed extraction (PerSeptiveBiosystems, Inc., Framingham, Mass.). The spectrum for peptideLysl56-Arg172 with mass 2097.04 is shown on the bottom panel (wild-typecells). The top panel shows the peptide resolved into two componentswhen isolated from cells bearing the T222P allele of the gene for ValRS.The second component has a mass of 2083.04, exactly 14 mass units lessthen the “wild-type peptide”. Multiple peaks correspond to ¹³C isotopicforms that separate peptides differing by 1, 2, 3, etc. mass units.

[0026]FIG. 3. A graph showing the results of growth of the valineauxotroph CU505 in the presence of a limiting supply of valine andincreasing concentrations of Abu. Overnight cultures of Δilv auxotrophscontaining the WT va/s (É) or the mutant va/S:T222P allele (J) and grownin MS glucose medium with limiting valine (0.04 mM Ile-Val. 0.3 mMIle-Leu) were diluted 1/1, the Val concentration was adjusted to 0.02 mMand the biomass was determined by measuring the optical density at 600nm after 24 h of growth at 30° C.

[0027]FIG. 4. A graph showing hydrolysis of valyl-tRNAile by wild typeand mutated IleRS. This figure shows the large decrease of the editingactivity of the mutant IleRS having 11 alanine residues in position aa239-250 compared with the editing activity of the wild type IleRS.(square; ▪):IleS ala 11 (mutant IleRS having 11 alanine residues inposition aa 239-250); (diamond, ♦): IleS wt (wild type IleRS)

[0028] FIGS. 5A and 5B: Correlation of Abu toxicity phenotypes andeffectiveness of deacylation by mutant enzymes.

[0029]FIG. 5A: Noncognate amino acid toxicity phenotypes. ValS nullstrains harboring each of the ValRS mutant enzymes in trans were platedon minimal medium. Abu (alpha-aminobutyrate) was loaded into a centralwell and plates were incubated for 24 hours at 42 deg. C. Degree oftoxicity in response to Abu was evaluated by the diameter of the regionof inhibited cell growth. Strains bearing the K277Q and T222P mutantvalS alleles showed the most severe response to Abu. The growth of D230Nvals was inhibited by high concentrations of Abu but the effect was notobserved at lower concentrations. The V276A mutation introduces a slightsensitivity to Abu. Similar phenotypes were observed in response toexogenous threonine (data not shown).

[0030]FIG. 5B: Histogram representation of the inhibition zone diametersof DvalS::kanR strains harboring each of the ValRS editing mutants onplasmid in response to exogenous Abu. Effects range from mild to severe.Histogram representation of the percentage of mischarged Thr-tRNAValremaining after incubation with 2 nM enzyme for 15 minutes at roomtemperature. Both show a distinct range in the severity of editingdefects caused by these point mutations. Taken together this illustratesa correlation between the ability of the mutant enzyme to deacylatemischarged amino acids from tRNAVal in vitro and the degree of in vivotoxicity.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention provides a method to diversify the chemicalcomposition of proteins produced in vivo by a cell comprising the stepof disabling the editing function of one of its aminoacyl tRNAsynthetases.

[0032] The phrase “a step of disabling the editing function of anaminoacyl tRNA synthetase”, is intended to designate a step throughwhich a mutant strain is obtained wherein an allele coding for anaminoacyl tRNA synthetasecomprises a mutation in its editing site ordomain resulting in:

[0033] the loss of the role of the editing function in restricting thegenetic code to 20 amino acids;

[0034] the loss of the role of the editing function in preventing theinvasion of noncognate amino acid; and/or

[0035] the misactivation of noncognate amino acids or/and the nonhydrolyzation of generated “noncognate amino acid-tRNA” normallyhydrolyzed by the editing function of the wild type cognate aminoacyltRNA synthetase,

[0036] these alterations of the editing function inducing the potentialmisincorporation of a noncognate amino acid, preferably a noncanonicalamino acid, in place of the cognate amino acid charged by the aminoacyltRNA synthetase encoded by the wild type allele.

[0037] For example, such a step of disabling the editing function of oneof an aminoacyl tRNA synthetase may comprise a step of:

[0038] selecting a mutant strain comprising an aminoacyl tRNA synthetasenatural variant having a mutation in its editing domain resulting tothese above cited editing function alterations; or preferably

[0039] selecting a mutant strain comprising an aminoacyl tRNA synthetasevariant having a mutation in its editing domain resulting to these abovecited editing function alterations, this variant being obtained bymutagenesis, such as by homologous recombination or replacement allelevector or by any mutagenesis methods known by the skilled person capableof introducing such a mutation, preferably integrated into the genome ofthe cell.

[0040] The term “cognate amino acid” as used in the presentspecification is intended to designate the amino acid normally chargedby the tRNA corresponding to (or associated with) the aminoacyl tRNAsynthetase wild type (for example: the cognate amino acid for valyl-tRNAsynthetase is valine).

[0041] By the term “cognate tRNA” as used in the present specificationis intended to designate the tRNA corresponding to (or associated with)the aminoacyl tRNA synthetase wild type (for example: the cognate tRNAfor valyl-tRNA synthetase is tRNAVal).

[0042] In an additional embodiment of the present invention, is provideda method for producing in vivo proteins comprising at least onenoncanonical amino acid comprising the step of:

[0043] a) selecting a cell strain wherein the editing function of one ofits aminoacyl tRNA synthetases has been disabled by mutagenesis, saiddisabled editing function allowing the aminoacyl tRNA synthetase tomischarge the cognate tRNA with said at least one noncanonical aminoacid;

[0044] b) culturing the selected strain in a culture medium comprisingsaid noncanonical amino acid, or one of its precursor, under favourableconditions for the growth of said strain; and

[0045] c) recovering from the culture medium or from the cells obtainedin step b) the proteins containing said noncanonical amino acid.

[0046] By “favourable growth conditions” is meant an environment that isrelatively favorable for cell growth and/or viability. Such conditionstake into account the relative availability of nutrients and optimaltemperature, atmospheric pressure, presence or absence of gases (such asoxygen and carbon dioxide), and exposure to light, as required by theorganism being studied.

[0047] By “precursor” is meant a compound which can be efficientlyconverted in vivo by the cell in said noncanonical amino acid.

[0048] In a preferred embodiment, the cell strain wherein the editingfunction of one of its aminoacyl tRNA synthetases has been disabled,comprises a mutation in the DNA sequence encoding the editing domain ofsaid disabled aminoacyl tRNA synthetase compared to the wild typeaminoacyl tRNA synthetase coding sequence.

[0049] In a more preferred embodiment, said DNA mutation leads to anamino acid mutation, preferably an amino acid substitution, morepreferably an amino acid single substitution, in the editing domain ofsaid aminoacyl tRNA synthetase.

[0050] In another preferred embodiment is provided a method according tothe present invention, wherein the disabled aminoacyl tRNA synthetase iscapable of mischarging its cognate tRNA with a canonical amino acidsterically similar to the amino acid charged by the wild type aminoacyltRNA synthetase on its cognate tRNA.

[0051] In another preferred embodiment is provided a method according tothe present invention, wherein the disabled aminoacyl tRNA synthetase iscapable of mischarging its cognate tRNA with a noncanonical amino acidsterically similar to the amino acid charged by the wild type aminoacyltRNA synthetase on its cognate tRNA.

[0052] In another aspect of the present invention, there is provides amethod for obtaining cells capable of producing in vivo proteinscomprising at least one noncanonical amino acid comprising the step ofmutagenizing the DNA sequence encoding the editing domain of anaminoacyl tRNA synthetase in a cell, said mutagenizing leading to anaminoacyl tRNA synthetase variant having an amino acid mutation in itsediting domain and said mutation allowing the aminoacyl tRNA synthetasevariant to mischarge its cognate tRNA with said at least onenoncanonical amino acid.

[0053] In preferred embodiment, there is provides a method for obtainingcells capable of producing in vivo proteins comprising at least onenoncanonical amino acid according to the present invention, comprisingthe steps of:

[0054] a) assaying the ability of an aminoacyl tRNA synthetase variantmutated in its editing domain for its ability to mischarge its cognatetRNA with a noncanonical amino acid;

[0055] b) mutagenizing the DNA sequence encoding the editing domain ofsaid aminoacyl tRNA synthetase in a cell, said mutagenizing leading toreplace the allele encoding the wild type aminoacyl tRNA synthetase byan allele encoding the aminoacyl tRNA synthetase variant assayed in stepa) and capable of producing detectable noncanonical amino acidmischarging;

[0056] c) optionally, identifying, selecting and/or cloning the cellscontaining such aminoacyl tRNA synthetase variant having the ability tomischarge one noncanonical amino acid.

[0057] In a preferred embodiment, the cell wherein the editing domain ofthe aminoacyl tRNA synthetase has been disabled, preferably bymutagenesis, is a microbial or animal cell, preferably a bacterium, ayeast or a fungus, more preferably a bacterium such as Escherichia colior Acinetobacter.

[0058] In another preferred embodiment of the present invention, isprovided a method, wherein the editing domain of the aminoacyl tRNAsynthetase of the target cell has been disabled by by homologousrecombination or by recombination into the genome of the target cellusing an allelic replacement vector.

[0059] The oligonucleotides comprising the nucleic fragment encoding themutated editing site, or portion thereof, and which contain the mutationto be introduced into the wild type allele of the target cell, can bechemically synthetized or synthetized by Polymerase Chain Reaction(PCR).

[0060] By microbial or animal cells are preferred cells that undergohomologous recombination. Such cells may be of bacterial, mycobacterial,yeast, fingal, algal, plant, or animal origin.

[0061] By “homologous recombination” is meant a process by which anexogenously introduced DNA molecule integrates into a target DNAmolecule in a region where there is identical or near-identicalnucleotide sequence between the two molecules. Homologous recombinationis mediated by complementary base-pairing, and may result in eitherinsertion of the exogenous DNA into the target DNA (a single cross-overevent), or replacement of the target DNA by the exogenous DNA (a doublecross-over event).

[0062] By “allelic replacement vector” is meant any DNA element that canbe used to introduce mutations into the genome of a target cell byspecific replacement of a native gene with a mutated copy. For example,gene replacement in bacteria is commonly performed using plasmids thatcontain a target gene containing a mutation and a negative selectablemarker outside of the region of homology. Such a plasmid integrates intothe target chromosome by homologous recombination (single cross-over).Appropriate selection yields cells that have lost the negative selectionmarker by a second homologous recombination event (double cross-over)and contain only a mutant copy of the target gene.

[0063] In another embodiment, the cell wherein the editing domain of theaminoacyl tRNA synthetase has been disabled or mutagenized may contain aselectable marker gene for identifying and selecting the cellscontaining the mutagenized DNA. The identification and selection of thatcells wherein the editing domain of the aminoacyl tRNA synthetase hasbeen disabled or mutagenized may be based upon the ability of the cellsto grow on selective medium, wherein a cell containing the selectablemarker can grow on selective medium, and a cell lacking this selectablemarker cannot grow, or grows more slowly, on selective medium.

[0064] In still another embodiment, the cell wherein the editing domainof the aminoacyl tRNA synthetase has been disabled or mutagenized maycontain a reporter gene, for identifying and selecting the cellscontaining the mutagenized DNA. The identification and selection of thatcells wherein the editing domain of the aminoacyl tRNA synthetase hasbeen disabled or mutagenized may be based on a reporter gene assay,wherein the expression by the cell of the reporter gene confirms thedisabling or the mutagenesis and a cell lacking the disabling or themutagenesis does not express the reporter gene.

[0065] By “selectable marker” is meant a gene that alters the ability ofa cell harboring this gene to grow or survive in a given growthenvironment relative to a similar cell lacking the selectable marker.Such a marker may be a positive or negative selectable marker. Forexample, a positive selectable marker (e.g., an antibiotic resistance orauxotrophic growth gene) encodes a product that confers growth orsurvival abilities in selective medium (e.g., containing an antibioticor lacking an essential nutrient). A negative selectable marker, incontrast, prevents cells harbouring this gene from growing in negativeselection medium, when compared to cells not harbouring this gene. Aselectable marker may confer both positive and negative selectability,depending upon the medium used to grow the cell. The use of selectablemarkers in prokaryotic and eukaryotic cells is well known by those ofskill in the art.

[0066] By “reporter gene” is meant any gene which encodes a productwhose expression is detectable and/or quantitatable by immunological,chemical, biochemical, biological, or mechanical assays. A reporter geneproduct may, for example, have one of the following attributes, withoutrestriction: fluorescence (e.g., green fluorescent protein), enzymaticactivity (e.g., lacZ/.beta.-galactosidase, luciferase, chloramphenicolacetyltransferase, alkaline phosphatase), toxicity (e.g., ricin), or anability to be specifically bound by a second molecule (e.g., biotin or adetectably labelled antibody). It is understood that any engineeredvariants of reporter genes, which are readily available to one skilledin the art, are also included, without restriction, in the foregoingdefinition.

[0067] By “identifying cells containing mutagenized DNA” is meantexposing the population of cells transformed with the mutagenized DNA toselective pressure (such as growth in the presence of an antibiotic orthe absence of a nutrient) consistent with a selectable marker carriedby the cell containing the recombined mutagenized DNA (e.g., anantibiotic resistance gene or auxotrophic growth gene known to thoseskilled in the art). Identifying cells containing the recombinedmutagenized DNA may also be done by subjecting transformed cells to areporter gene assay. Selections and screens may be employed to identifycells containing the recombined mutagenized DNA, although selections arepreferred.

[0068] In another aspect, the present invention is directed to a methodfor selecting an aminoacyl tRNA synthetase variant capable ofmischarging its cognate tRNA with a noncognate and/or a noncanonicalamino acid, comprising the steps of:

[0069] a) elaborating a DNA construct comprising a DNA sequence encodingan aminoacyl tRNA synthetase variant wherein said aminoacyl tRNAsynthetase variant has at least an amino acid mutation, preferably asubstitution, more preferably a single substitution, in its editingdomain compared with the wild type aminoacyl tRNA synthetase;

[0070] b) transforming a host cell with the DNA construct of step a)and, after a step of culturing said transformed host cell, recoveringand, optionally, purifying the recombinant aminoacyl tRNA synthetasevariant expressed by the host cell;

[0071] c) assaying the ability of the recombinant aminoacyl tRNAsynthetase variant recovered in step b) for its ability to mischarge itscognate tRNA with a noncognate and/or a noncanonical amino acid; and

[0072] d) selecting said aminoacyl tRNA synthetase variant if detectablemischarging has been produced in step c).

[0073] Such methods for assaying the ability of the recombinantaminoacyl tRNA synthetase variant recovered in step b) for its abilityto mischarge its cognate tRNA with a noncognate and/or a noncanonicalamino acid, are disclosed for example in the following examples (seeFIG. 2B). The methods known to those skilled in the art for assayingsuch an ability may be used.

[0074] By “transformation” is meant any method for introducing foreignmolecules, such as DNA, into a cell. Lipofection, DEAE-dextran-mediatedtransfection, microinjection, protoplast fusion, calcium phosphateprecipitation, retroviral delivery, electroporation, naturaltransformation, and biolistic transformation are just a few of themethods known to those skilled in the art which may be used.

[0075] In another aspect, the invention provides an isolated aminoacyltRNA synthetase variant capable of mischarging its cognate tRNA with anoncanonical amino acid, obtainable by the method for selecting anaminoacyl tRNA synthetase variant according to the present invention.

[0076] The isolated nucleic acid sequence encoding the aminoacyl tRNAsynthetase variant according to the invention or vectors comprising anucleic acid encoding said aminoacyl tRNA synthetase variant form alsopart of the present invention.

[0077] In another aspect, the invention provides a transformed cellcomprising a nucleic acid encoding an aminoacyl tRNA synthetase variantaccording to the present invention.

[0078] In a preferred embodiment, said transformed cells arecharacterized in that said nucleic acid encoding an aminoacyl tRNAsynthetase variant according to the present invention is integrated inthe genome of said cell.

[0079] In a more preferred embodiment, said transformed cells arecharacterized in that the nucleic fragment comprising the mutation,preferably a substitution, more preferably a single substitution,leading to the alteration of the editing function of the aminoacyl tRNAsynthetase variant, has been integrated into the genome of saidtransformed cell by using mutagenesis, such as homologous recombination,replacement allele vector or any mutagenesis methods for introducingnucleic acid molecules such as DNA, into a cell, known to those skilledin the art, such as lipofection, DEAE-dextran-mediated transfection,microinjection, protoplast fusion, calcium phosphate precipitation,retroviral delivery, electroporation, natural transformation andbiolistic transformation.

[0080] The invention also relates to isolated prokaryotic or eukaryoticcells capable of producing a protein the amino acid sequence of whichcomprises at least one noncanonical amino acid, characterized in thatthey comprise an aminoacyl-tRNA synthetase variant which is capable ofcharging onto one of its cognate tRNAs a noncanonical amino acid or anamino acid other than the cognate amino acid, and in that the nucleicacid sequence of the allele encoding said aminoacyl-tRNA synthetasevariant includes at least one mutation, preferably a substitution, morepreferably a single substitution, compared with the sequence of thecorresponding wild-type allele, said at least one mutation integratedinto the genome being located on the editing site of said aminoacyl-tRNAsynthetase and having been introduced by a technique of mutagenesis,such as genetic recombination.

[0081] In another aspect, the invention also comprises the use of atransformed cell according to the invention for producing protein, inparticular recombinant protein, the amino acid sequence of whichcomprises at least one unconventional amino acid.

[0082] In a preferred embodiment, the present invention is directed to amethod for the production of proteins comprising a noncanonical aminoacid characterized in that said method comprises the following steps:

[0083] a) culturing a transformed cell comprising an allele encoding anaminoacyl tRNA synthetase variant according to the invention in aculture medium containing a noncanonical amino acid capable of beingmischarged by the cognate tRNA of the aminoacyl tRNA synthetase variantcontained in said cell, in a culture medium and under culture conditionswhich allow the growth of said cell; and

[0084] b) recovering and, optionally, purifying the proteins comprisingsaid noncanonical amino acid from the culture medium (supernatant) orfrom the cells (cells pellet) of step a).

[0085] Proteins comprising a noncanonical amino acid obtained by theabove method according to the invention form also part of the presentinvention.

[0086] The processes for purifying protein, which may be natural orrecombinant, conventionally used by those skilled in the art generallyemploy methods used individually or in combination, such asfractionation, chromatography methods, immunoaffinity techniques usingspecific mono- or polyclonal antibodies, etc.

[0087] The presence of a noncanonical amino acid on the protein to bepurified, which noncanonical amino acid could have a specific functionalgroup, may facilitate its purification by reacting selectively with thepurification support without modifying the activity of the protein.

[0088] Among the proteins which can be produced by a process accordingto the invention, mention may be made, but without being limitedthereto, of proteins which, through the incorporation of at least onenoncanonical amino acid, make it possible to obtain a desired activitywhich a protein the sequence of which includes only canonical aminoacids does not make it possible to obtain. The term “activity” isintended to refer, in general, to any activity such as a physiologicalor biological activity, even partial, relating to unicellular ormulticellular organisms, such as for example a structural or biochemicalactivity, for example an enzymatic or antigenic activity, an activity ofantibody type, or an activity which modulates, regulates or inhibitsbiological activity, or such that it allows the implementation thereofin a process for biosynthesizing or for biodegrading chemical orbiochemical compounds.

[0089] Among the proteins which can be produced by a process accordingto the invention, mention may also be made of proteins for which theincorporation of at least one noncanonical amino acid is carried outsuch that there results therefrom no substantial modification of thebiological activity of the corresponding unmodified protein. Besides theconserved biological activity of the corresponding unmodified protein,these proteins according to the invention will have a noncanonical aminoacid with specific properties which may be advantageously exploited.

[0090] Among the specific properties conferred by the presence of anoncanonical amino acid, mention may be made in particular of theproperties linked to the presence of a functional group on saidnoncanonical amino acid, capable of reacting easily and specificallywith a chemical or biochemical compound under conditions which make itpossible not to modify the activity of the protein or which avoidmodifying the conventional amino acids.

[0091] The presence of this specific functional group may advantageouslybe used, for example, for:

[0092] (i) purifying any protein, in particular any recombinant protein,which incorporates said unconventional amino acid;

[0093] (ii) coupling such a protein to a solid support;

[0094] (iii) coupling to such a protein molecules capable of beingdetected, such as spectroscopic probes of varied nature;

[0095] (iv) coupling to such a protein lipophilic or hydrophilicpolymers which allow the solubilization thereof in solvents or whichallow masking against recognition by antibodies;

[0096] (v) coupling such a protein to a polynucleotide;

[0097] (vi) coupling such a protein to a chemical or biochemicalcompound the presence of which makes it possible to increase, todecrease, to modify, to regulate or to target the biological activity ofsaid protein, or to modify the bioavailability thereof as a compound fortherapeutic use; or

[0098] (vii) permanently attaching to such a protein a coenzyme whichotherwise would diffuse in solution.

[0099] Also included in the present invention are the processesaccording to the invention, characterized in that said transformed cellaccording to the present invention comprises a homologous orheterologous gene of interest the coding sequence of which includes atleast one codon encoding an amino acid the cognate tRNA of which can bemischarged by the aminoacyl tRNA synthetase variant contained in saidtransformed cell.

[0100] In general, the homologous or heterologous gene of interest,which can be isolated by any conventional technique, such as cloning orPCR (Polymerase Chain Reaction), or chemically synthesized, may bechosen from genes encoding any protein which can be used as atherapeutic or cosmetic compound, or as a diagnostic reagent or as acompound which can be used in a biosynthesis or biodegradation process.The protein of interest can consist of a mature protein, a precursor,and in particular a precursor intended to be secreted and comprising asignal peptide, a truncated protein, a chimeric protein originating fromthe fusion of sequences of diverse origins, or a mutated protein havingimproved and/or modified biological properties.

[0101] The invention also comprises a process for producing a proteinaccording to the invention, characterized in that the culture medium ofstep a) also comprises the compounds required for inducing the synthesisof the protein encoded by said homologous or heterologous gene ofinterest. These compounds are known to those skilled in the art anddepend, in particular, on the cell and on the homologous or heterologousgene selected.

[0102] In any of the method according to the present invention, it ispreferred that said aminoacyl tRNA synthetase is an aminoacyl tRNAsynthetase selected from the group consisting of the aminoacyl tRNAsynthetase comprising an editing function corresponding to an editingsite or domain encoded by a portion of the DNA encoded said aminoacyltRNA synthetase, preferably encoded by a DNA portion having at leastconserved residues compared after alignment with the editing site of thevalyl-tRNA synthetase and isoleucyl-tRNA synthetase as shown in FIG. 2A,preferably selected from the group consisting of the aminoacyl tRNAsynthetase valyl-tRNA synthetase, isoleucyl-tRNA synthetase, leucyl-tRNAsynthetase, alanyl-tRNA synthetase, prolyl-tRNA synthetase,threonyl-tRNA synthetase, phenyl-tRNA synthetase and lysyl-tRNAsynthetase which are known to have an editing site or domain (see forIle RS Baldwin, A. N. and Berg, P. (1966) J. Biol. Chem. 241, 839-845and Eldred, E. W. and Schimmel, P. R. (1972) J. Biol. Chem. 247,2961-2964; for Val RS, Fersht, A. R. and Kaethner, M. M. (1976)Biochemistry. 15 (15), 3342-3346; for Leu RS, English, S. et al., (1986)Nucleic Acids Research. 14 (19), 7529-7539; for Ala RS, Tsui, W.-C. andFersht, A. R. (1981) Nucleic Acids Research. 9, 7529-7539; for Pro RS,Beuning, P. J. and Musier-Forsyth, K. (2000) PNAS. 97 (16), 8916-8920;for Thr RS, Sankaranarayanan, R. et al., (2000) Nat. Struct. Biol. 7,461-465 and Musier-Foryth, K. and Beuning, P. J. (2000) Nat. Struct.Biol. 7, 435-436; for PheRS, Yarus, M. (1972) PNAS. 69, 1915-1919 andfor LysRS, Jakubowski, H. (1997) Biochemistry. 36, 11077-11085.

EXAMPLES Example 1

[0103] A direct selection for restoring an enzymatic activity throughincorporation of Abu could not be easily set up because its aliphaticside chain lacks chemical reactivity and therefore cannot act as acatalytic residue. We thus resorted to an indirect scheme based on thestructural resemblance of Abu with Cys (FIG. 1A), an essential catalyticresidue in numerous enzymes. Selecting a synthetase mutant thatmischarged its cognate tRNA with Cys might result in Abu beingmischarged by the mutant synthetase.

[0104] We took advantage of the thyA conditional selection screen in E.coli, based on the absolute requirement for an active thymidylatesynthase when thymidine is not provided as a growth factor (13). Thissame screen was used previously to assess the potency of suppressorCys-tRNAs in codon misreading (14) and to enforce phenotypic suppressionby the non-canonical azaleucine (15). An entire set of plasmid-bornethya alleles with all 64 different codons at position 146 wasconstructed for altering the catalytic site occupied by an essential Cys(16, 17). Each allele was tested for its ability to restore growth to anE. coli strain (lacking the chromosomal copy of thyA) on mineral glucosemedium in the absence of thymidine (14, 15). Three of the 64plasmid-borne thyA alleles restored growth. These had one of threecodons-UGU, UGC, or UGA. The growth responses of the UGU and UGC alleleswere expected, as these code for cysteine. The positive response of UGA(a termination codon in E. coli) likely results from read-through byCys-tRNA, and thus demonstrates the sensitivity of the selection assay.

[0105] Strains bearing inactive alleles of thyA were then tested to seeif they could be suppressed by supplying them with excess L-cysteine inmineral medium devoid of thymidine. Shallow growth was reproduciblyobserved on L-cysteine gradient plates (18) for the missense alleleshaving any of the eight codons AUN and GUN alone. Growth was strongerwith alleles bearing any of the four Val codons (GUU, GUC, GUA, and GUG)than for those with the three Ile codons (AUU, AUC, and AUA) or for theMet codon AUG. Cysteine-suppression of the three Ile codon-bearingalleles and of the Met codon-bearing allele was abolished by addition ofexogenous L-isoleucine and L-methionine, respectively. Suppression ofthe four Val codon-bearing alleles was abolished by addition ofexogenous L-valine plus L-isoleucine but not by L-isoleucine alone (18).The four Val146 alleles gave a similar growth response in cysteinegradient plates, despite being decoded by three different tRNAValisoacceptors, thus suggesting that Cys is being mischarged onto allthree Val isoacceptor tRNAs by ValRS. Altogether, these resultssuggested that ValRS catalyzed the formation of Cys-tRNAVal in vivo at arate sufficient for active thymidylate synthase production and that thismischarging reaction was prevented by increasing the intracellularconcentration of L-valine. This interpretation is in line with earlierreports of Cys misactivation by ValRS in vitro (11).

[0106] Suppression of thyA:Val146 alleles was weak on plates andrequired high L-cysteine concentrations (developed from a gradientstarting at a concentration of 100 mM). It could thus be anticipatedthat a scarce L-cysteine supply should select for an enhanced efficiencyof phenotypic suppression. Two experimental procedures were followed tothis end. In the first procedure strain β5519(thyA::erm+AnrdD::kan+pTS13 (bla+thyA:146GUA) was propagated over 100generations in serial liquid culture with limiting cysteine (1.5 mM)under anaerobic conditions resulting in isolation of strain p5420 (19).The second procedure relied on a one-step selection under aerobicconditions on plates containing a non-oxidizable precursor that isinefficiently converted into cysteine by E. coli (S-carbamoyl-cysteine(Sec, FIG. 1A) (19)). A mutator marker (dnaQ or muts) was introducedinto the test strain P5519 to increase the frequency above 10⁻¹⁰ ofScc-suppressible thymidine auxotrophs (19). This approach resulted inthe isolation of four strains: β5456, β5479, β5485 and β5486. All fiveisolated strains were found not to grow at 42, in agreement with theheat sensitivity generally caused by translational errors (20). Thecysteine-suppression phenotype is shown for one of these strains (β5456)in FIG. 1B, together with its abolition by L-valine (supplied as anIle-Val peptide).

[0107] Judging from these phenotypes, mutations in the vals gene forvalyl-tRNA synthetase were suspected for each isolated strain. To testfor this possibility, we took advantage of the nrdD::kan+marker (19)located 0.4 min from vals on the E. coli chromosome. For the fivemutants, the trait of cysteine- or Scc-suppressible thymidine auxotrophywas indeed found to co-transduce with the nrdD::kan+marker in aproportion of about 45%. Further characterization of the valS mutationswas performed by PCR-amplification followed by sequencing of fivenrdD::kan+transductants exhibiting Scc-suppressible thymidine auxotrophyderived from the five isolated strains (21). As shown in Table 1, eachof the five mutants contained a single amino acid substitution atpositions within the conserved editing domain (known as CP1) of ValRS(22). The following mutations were identified: T222P, R223H, D230N,V276A, and K277Q. Remarkably, two of these positions (T222 and D230)align with conserved positions in IleRS that had been demonstratedpreviously to be involved in the hydrolytic editing of misacylatedVal-tRNA^(Ile) (23) (FIG. 2A). TABLE 1 Mutations of the valS geneselected by suppression of the thymidine auxotrophy of strain β5419(ΔthyA::erm⁺ ΔnrdD::kan⁺pTS13 (bla⁺thyA:146GUA)). Method of MutatorCodon of valS Strain isolation genotype 222 223 230 276 277 β5419 — wtACC CGT GAT GTG AAA Thr Arg Asp Val Lys β5456 Selection ΔdnaQ CCC CGTGAT GTG AAA on Scc Pro β5479 Selection ΔmutS ACC CAT GAT GTG AAA on SccHis β5486 Selection ΔmutS ACC CGT AAT GTG AAA on Scc Asn β5485 SelectionΔmutS ACC CGT GAT GCG AAA on Scc Ala β5520 Selection wt ACC CGT GAT GTGCAA on Cys under Gln anaerobiosis

[0108] ValRS is known to misactivate Thr and generate Thr-tRNA^(Val),which normally is hydrolyzed by the ValRS editing activity (9). If theValRS mutants in Table 1 are impaired for editing, then strains bearingeach of these mutations should misincorporate Thr into protein, andL-threonine would then be toxic in these strains. The growth of the fivedifferent vals strains was therefore tested in the presence ofL-threonine. All displayed high sensitivity towards exogenousL-threonine (at 2 mM), while the parent strain β5419 was insensitive toL-threonine at all concentrations. The results for the strain carryingthe valS:T222P allele are shown in FIG. 1C.

[0109] The phenotype of the valS:T222P allele suggested that the T222Penzyme mischarged tRNA^(Val) with Thr and Cys in vivo. The T222P mutantenzyme was, therefore, expressed and partially purified so that it couldbe directly assayed for the ability to misacylate tRNA^(Val). Thepurified enzyme had the same activity as the wild-type enzyme forcharging with valine (FIG. 2B, inset). In contrast, the T222P mutantenzyme misacylated tRNA^(Val) with Thr to give Thr-tRNA^(Val) while thewild-type enzyme produced no detectable mischarged tRNA^(Val) (FIG. 2B).Misacylation of tRNA^(Val) with cysteine was also catalyzed only by themutant enzyme.

[0110] We anticipated that ValRS mutants that misincorporated Cys wouldalso misincorporate Abu. Indeed, the strain carrying the valS:T222Pallele on the chromosome (p5456) was sensitive to Abu (FIG. 1C) (whereasits wild-type counterpart was not), suggesting incorporation of Abu inresponse to Val codons. With this in mind, we showed that Abu couldcontribute to the relief of L-valine auxotrophy of the Δilv strainCU505, but only in the presence of the valS:T222P allele (19). Wholecell protein was isolated from strain CU505 grown in the presence of Abu(0.2 mM), in the presence or absence of the valS:T222P allele in thehost cell. Analysis of amino acid composition showed that 24% of thevaline was replaced by Abu only in the strain harboring the mutantallele (Table 2). Finally, the valine-rich yeast protein AlaXp(swissprot:P53960) was overexpressed and purified from strainscontaining the valS:T222P allele grown in the presence of Abu. Theprotein samples were digested by trypsin and analyzed by massspectrometry. MALDI analysis showed that, when AlaXp was produced in thestrain carrying the T222P mutation, it contained a mixture of Val andmisincorporated Abu (FIG. 2C). For a given peptide the degree ofmisincorporation ranged between 9.5% and 18% per Val codon. Sequencingof several Abu-containing peptides confirmed that Abu was specificallymisincorporated into positions designated by Val codons.

[0111] Table 2. Incorporation of Abu into cells bearing wild-type andT222P mutant alleles of valS. Cultures of the Δilv strain CU505 (19) andthe isogenic strain P5498 carrying the valS:T222P allele were grownovernight in minimal medium (27) with Ile-Leu (0.3 mM) and limitingvaline (0.04 mM Ile-Val), diluted (½), and adjusted with Ile-Val (0.02mM) with or without Abu (0.2 mM). After 24 hours of growth, totalprotein was extracted as follows. Cells were first harvested bycentrifugation and washed in cold 10% trichloroacetic acid (TCA, ½ ofthe culture volume). Cells were then re-centrifuged at 4000×g for 10min, resuspended in cold 10% TCA ({fraction (1/10)} of the culturevolume) and centrifuged again. The washed cells were resuspended in 5%TCA, heated at 95° C. for 30 minutes and centrifuged (4000×g, 10 min).The precipitate was washed three times with cold acetone and dissolvedin 50 mM NH₄HCO₃. Proteins were hydrolyzed in 6 N HCl-0.2% phenol at110° C. for 20 h in sealed tubes. Norleucine was added as an internalstandard. Aliquots of the hydrolysates were analyzed on a Beckman 6300Amino Acid Analyzer. Amino acids were quantified by appropriatestandards and values are presented relative the wild-type (WT) controlthat lacked Abu. Amino acid incorporated WT T222P WT + Abu T222P + AbuAbu 0.0 0.0 0.0 0.24 Val 1.0 0.95 1.0 0.73 Val ± Abu 1.0 0.95 1.0 0.97Ile 1.0 1.0 1.0 1.0 Ala 1.0 0.97 1.0 0.92

Example 2

[0112] Directed evolution scheme. To avoid a decrease of cysteineconcentration in the medium due to oxidation, the selections werecarried out under strictly anaerobic conditions. The biomass of cells incultures relative to the cysteine concentration (measured as opticaldensity at 600 nm after 24 h of growth at 30° C.) gradually increasedfour-fold when the thyA strain β5366 (AthyA::erm⁺ pTS13 (bla⁺thyA:146GUA)) was propagated by serial transfer in mineral glucosemedium supplemented with 1.5 mM cysteine. A high concentration ofcysteine was required because the input population could hardly bepropagated otherwise. After 16 inoculations, each at a dilution of{fraction (1/100)} (a total of about 100 generations), single colonieswere isolated on mineral glucose plates supplemented with thymidine (arepresentative of which was designated strain β5520).

[0113] One-step selection scheme. For the one-step selections, cysteineoxidation under aerobiosis and subsequent cystine precipitation from thegrowth medium was avoided by use of the non-oxidizable precursorS-carbamoyl-cysteine (Scc). Scc is a poor precursor of cysteine andsustains growth of a Cys-auxotrophic E. coli strain less efficientlythan cysteine. However, at concentrations above 1 mM, Sce gave rise tosuppression of the thymidine auxotrophy of strain β5520, while in theabsence of thymidine no growth was detectable below 1 mM Scc. When 35419cells bearing the thyA:Val146GUA allele on a high copy plasmid wereplated on mineral glucose plates supplemented with Scc (3 mM), nocolonies grew after prolonged incubation at 30° C. (The experiment wasdesigned to detect a mutant at a frequency of 10⁻¹⁰.) To increase themutation frequency, a mutS::spc+ (33) mutator allele was introduced inthe genetic background of strain 135419 by P1 transduction, yieldingstrain β5432 (the mutS::spc+ disruption disables the mismatch repairsystem and leads to random transitions and frameshifts (34)). Coloniesthen appeared on the same medium with a frequency of about 10⁻⁸. Nocolonies were found on plates lacking Scc. A comparable frequency ofScc-suppressible clones was obtained after introduction by P1transduction (33) of a dnaQ mutator allele (33) (to give strain 135435).

[0114] Abu misincorporation. The valine auxotroph CU505 (33) was grownin the presence of a limiting supply of valine and increasingconcentrations of Abu (FIG. 3). The biomass of cells in culturesrelative to the valine concentration in the medium did not change up toa 1 mM concentration of Abu. In contrast, when the valST222P allele wasintroduced into the chromosome (strain β5498), the yield of cells wasdiminished in the absence of Abu but increased up to 30% when Abu (0.2mM) was added.

[0115] Thus, E. coli strains that proliferate only because ofinfiltration of the Val coding pathway were selected and all containedmutations leading to single amino acid substitutions in the editing siteof ValRS. This observation is consistent with a central role for editingin restricting the genetic code to twenty amino acids, by preventing theinvasion of other amino acids such as Abu. Indeed, the editing sites inIleRS and ValRS are rigorously conserved in even the most deeplybranched organisms in the tree of life. However the translation accuracymaintained by editing may prevent further chemical diversification ofproteins. Thus, disabling the editing function of a synthetase, asdemonstrated in the present work, offers a a powerful approach todiversify the chemical composition of proteins produced in vivo.

[0116] For some synthetases, accuracy depends critically on an editingfunction at a site distinct from the aminoacylation site. Mutants ofEscherichia coli that mischarge tRNA^(Val) with cysteine were sought byrandom mutagenesis of the whole chromosome. All mutations obtained werelocated in the editing site of valyl-tRNA synthetase. Over 20% of thevaline in cellular proteins from such an editing mutant organism couldbe replaced with the noncanonical aminobutyrate, sterically similar tocysteine. Thus, the editing function may have played a central role inrestricting the genetic code to twenty amino acids. Disabling thisediting function offers a powerful new approach for diversifying thechemical composition of proteins and for emulating ambiguousevolutionary stages of translation.

Example 3 Correlation Between Toxicity of α-Aminobutyrate for E. coliStrains Harboring Different valS Alleles Mutated in the Editing Domainand Results of in Vitro Editing Assays Carried Out with theCorresponding ValRS Enzyme Variants

[0117] To investigate more deeply the editing phenotypes of the fiveaforementioned mutant ValRSs, and the relationship between cellviability and editing, plasmids harboring genes for mutant and wild-typeValRSs were constructed and placed under control of anarabinose-inducible promoter. These constructs were then used toinvestigate the in vivo phenotypes of the mutant enzymes. Separately,His-tagged versions of the mutant enzymes were constructed in parallelfor use in purifying the enzymes for studies of aminoacylation andediting activities in vitro.

[0118] Construction of vals knockout strains: A 1.7 kb portion of thevalS gene from pET16valS that contained the region encoding the editingdomain of ValRS was excised using SalI and XhoI. This region was thenreplaced with a kanamycin marker (1.2 kb in size) liberated from pUC4Kby flanking SalI restriction sites. Overhanging regions of the valS geneof 406 and 708 base pairs respectively were left to allow for homologousrecombination (resulting plasmid named pLAN362). The kanamycin markercontained its own promoter and was inserted in the forward orientationwith respect to the valS gene. To increase the amount of linearizedproduct used for homologous recombination, the valS::kan from pLAN362was PCR-amplified using primers annealing to the T7 promoter andterminator. The products of this reaction were visualized and purifiedin a 1% agarose TAE gel.

[0119] To get recombination into the E. coli chromosome, the linearizedvalS::kan was transformed by electroporation into a hyper recombinantstrain of E. coli (JC8679, Stewart et al.) containing plasmid-bome valSfrom Haemophilus influenzae (pSU18valS). Recombinants were selected onLuria Broth agar plates supplemented with chloramphenicol (strain JC8679is CmR) and low kanamycin (25 ug/ml) because efficiency of kanR markerscan be decreased when it recombines into the chromosome, one recombinantchosen and named strain PS2838. PS2838 was infected with a PI phagestock, phage were allowed to propagate for 3 hours then were stored inCHCl3 at 4C. Transfer of genes and resistance markers was done by P1phage transduction using standard protocols (Miller 1972) into MG1655wild-type E. coli containing each of the valSpBAD constructs expressingeither wild-type ValRS, T222P, R223H, D230N, V276A, and K277Q.Transductants were selected by plating on Luria Broth agar platessupplemented with kanamycin (25 ug/ml) and 0.2% arabinose, incubating at25C for 48 hours.

[0120] As expected transduction of the MG1655 strain alone did not yieldcolony growth. All transductants were restreaked onto media supplementedwith 50 ug/ml kanamycin to ensure kanR and insertion of kanamycin markerinto the E. coli chromosomal valS gene was verified by amplifying with aforward primer that annealed to a region on the chromosome just upstreamof the valS gene (valS. 103) and a reverse primer annealing to themarker itself (puc4k.o14). Transduction yielded the following strains ofvalS::kanR: wt ValRS-PS2847, T222P-PS2849, R223H-PS2862, D230N-PS2865,V276A-PS2851, K277Q-PS2853.

[0121] Plasmid Construction: A cautious approach was taken in thisconstruction due to a lack of success introducing valS editing mutantspreviously. With this in mind initial constructs were done in avariation of pBAD 18, a plasmid allowing for tight regulation ofexpression under control of an arabinose inducible promoter. Parentplasmid was a Histidine tagged vals construction received from JackHorowitz which we verified by sequence analysis to done be pET16b(Novagen). Utilizing an internal restriction site and a restriction sitelocated downstream of the valS gene in the multiple cloning site ofpET16b, a large BamHI fragment was subcloned into pUC19 (pSC02). Inparallel, the parent plasmid was digested with SalI and BglII,restriction sites that flank the region of valS encoding the CP1, andthis small segment was ligated into pUC19 (pSCO5).

[0122] The QuikChange mutagenesis kit (Stratagene, La Jolla, Calif.) wasused on pSCO5 to introduce an A to C base change at nucleotide 663 toachieve the Thr to Pro substitution in residue 222 of ValRS. Both thewild-type and T222P SalI-BglII fragments were subcloned into pSCO2. Theresulting vectors were digested with NcoI and SmaI to release fragmentsof the entire downstream region of valS which were then cloned intopVDC441, a variation of pBAD 18 into which the upstream portion of valSfrom the pET16bvalS plasmid had been cloned using EcoRI and KpnI. TheHis tagged T222P construct was constructed by subcloning the mutatedregion of valS (DraI/XhoI) from pVDC447 and ligating it into thepET16bvalS. The wild-type valS constructs, pBAD 18valS and pET16bvalS,were eventually used as templates in the QuickChange mutagenesisprotocol to generate mutations in valS corresponding to the mutantsR223H, D230N, V276A, and K277Q. The entire valS gene for these vectorswas then sequenced for each of these vectors to verify their integrity.

[0123] Mutant Toxicity Response to High Levels of α-Aminobutyrate:ΔvalS::kan+ strains expressing wild-type ValRS, T222P, R223H, D230N,V276A, and K277Q were each isolated on mineral standard medium (MS)(Richaud et al, 1993) supplemented with 0.2% glycerol, 0.02% arabinoseand ampicillin (100 ug/mL). Single colonies from each strain wereinoculated into liquid cultures of the same media composition and grownovernight to saturation. Cells were diluted in media 1:100 and a lawn ofcells was spread and allowed to dry onto MS medium plates.α-aminobutyrate (50 uL of 0.1M) was added to a central well created ineach plate and plates were then incubated for 24 hours at 42C. Therelative response of each strain to high levels of noncognateα-aminobutyrate could then be evaluated based on diameter of toxicityhalo observed.

[0124] Expression and Purification of ValRS Proteins: pET16b plasmidscorresponding to the wild-type ValRS and each of the ValRS mutants weretransformed into BL21 competent cells (Novagen). These cells werecultured in 250 mL of Luria Broth supplemented with ampicillin (100ug/mL), when cells reached an optical density at 600 nm of 1.0expression of ValRS was induced with 1 mM IPTG for 5 hours. Cells werethen stored at −80C until purification. Cells were resuspended (50 mMNa₂PO₄, 300 mM NaCl, 50 mM B-ME, 30 mM Imidazole pH 7.4) and lysed 2× inFrench Press. Lysates were bound a Ni-NTA affinity column and eluted inlysis buffer with a gradient ranging from 30 mM to 250 mM Imidazole.Collected fractions were visualized on an 8% SDS-polyacyrlamide gelstained with coomassie brilliant blue to ensure purity, purest fractionswere pooled and dialyzed into 25 mM Tris-HCl, 1 mM B-ME pH 7.5. Enzymeconcentrations were determined by Bradford assay.

[0125] Aminoaclyation Assay, Misacylation, and Deacylation:Aminoacylation assays were performed at 37C in a 100 uL volumecontaining buffer (20 mM HEPES, 0.1 mM EDTA disodium salt, 0.15M NH₄Cl,10 ug/mL BSA pH 7.5), 2 M MgCl₂, 0.7 μM [³H]Val, 20 μM cold Val, 2 μMtRNA_(val) (Sigma) and 20 nM ValRS enzyme (adapted from Hendrickson etal. 2000). Aliquots (10L) of the reaction mixture were precipitated withtricholoracetic acid, and the level of aminoacylation of the tRNA wasdetermined by scintillation counting.

[0126] Misacylation of Thr onto tRNA_(val) was performed in the sameconditions except 5.86 μM [³H]Thr was substituted forVal in thereaction. To test deacylation rates for these enzymes it was necessaryto generate [³H]Thr-tRNA_(val). This was done using ValRS with the T222Psubstitution purified previously (Doring et al, 2001), this enzyme wasshown to form the [³H]Thr-tRNAval complex. Enzyme was incubated with 2.5μM tRNA_(val) and 45 uM [³H]Thr, incubated at 37C for 45 minutes,extracted twice in phenol:chloroform, and ethanol precipitated. Pelletwas dissolved in 100 μL sterile H₂O and scintillation counting was usedto determine the success of the reaction. Deacylation reactions, done intriplicate for each enzyme, were performed in 150 mM Tris-HCl (pH 7.5),100 μg/ml BSA, 10 mM MgCl2. At room temperature 2 nM enzyme was combinedwith [³H]Thr-tRNA_(val), at 3, 6, 9, 16, and 30 minutes aliquots (9 μL)of the reaction mixture were precipitated with tricholoracetic acid, andthe amount of misaminoacylated [3H]Thr-tRNA_(val) remaining in thesample was determined by scintillation counting. A no enzyme controlreaction was included to provide reference point.

[0127] When the percentage of Thr-tRNA^(Val) hydrolyzed by each enzymein deacylation assays in vitro was compared to the observed Abu-inducedinhibition zone diameters in vivo, a pattern was clear (FIG. 5A). TheT222P and K277Q ValRS mutant proteins have the most severe defects, forexample, in deacylation of Thr tRNA^(Val) in vitro (FIG. 5B) Strainsbearing these mutations show a toxic response to only slightly raisedlevels of Abu in vivo. These two enzymes also accumulated high levels ofmischarged Thr-tRNA^(Val) in vitro. The D230N mutation appears to beless detrimental to the deacylase activity, exhibiting a slowed rate ofdeacylation. Similarly, toxicity in vivo was limited to intermediatelevels of Abu. Having the V276A mutation appears to impart the leasttoxic response to exogenous Abu. This phenotype is reflected in adeacylation rate that is closer to wild-type ValRS than to that of theother mutant enzymes. Both D230N and V276A ValRS showed low levels ofmischarging with respect to the other mutant enzymes as well. Thus, theability of the mutant enzymes to hydrolyze in vitro misaminoacylatedamino acids from tRNA^(Val) correlated with the toxicity in vivo ofexogenous noncognate amino acids that were added to cells bearing thesame mutations in their ValRS

Example 4 Construction of Strains of E. coli Expressing anIsoleucyl-tRNA Synthetase Mutated in the Editing Site

[0128] Several studies have identified the editing domain of IleRS(Schmidt at Schimmel, 1004, Science), and more particularly the aminoacids of the 239 to 250 region which are extremely conserved in theIleRS sequences of different organisms. Point mutations in this regiongenerate enzymes partially or totally deficient in the editing function(Hendrickson, 2000). An artificial allele of the gene iles coding forisoleucyl-tRNA E. coli synthetase containing a succession fine residuesreplacing residues 239 to 250 has been constructed in the followingmanner: the the pVDC433 (Hendrickson et al. 2001), derived from plasmidpBAD (Guzman et al., 1995) by insertion of the wild type ileS gene isdigested by restriction enzymes ClaI and SpeI according to theinstructions of the supplier, thus eliminating nucleotides 717 to 750 ofthe gene.

[0129] The 5′ phosphorylated oligonucleotides (Invitrogen LifeTechnologies): 5′pCTAGTAATCGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGCGCGC AATATand 5′pCGATATTGCGCGCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCGA TTA

[0130] are hybridized and ligated with the plasmid pVDC433, previouslycut by SlaI and SpeI under the following conditions: 0.3 pmoles ofvector and 3 pmoles of each oligonucleotide are incubated for 10 minutesat 70 degrees C. after precipitation and resuspension in 10 ul of H₂O,after return to room temperature; the ligation is carried out usingstandard protocols. The ligation mixture is utilized to transformcompetent cells of the SureTM strain (Stratagene) by electroporationfollowing the protocol of the supplier. The transformants are obtainedand plasmid DNA prepared (Qiaprep Kit, Qiagen). The plasmid obtained inthis manner, pTLH33, is sequenced and the insertion of 11 alanine codonsis verified.

[0131] The pTLH33 plasmid is inserted in the wild type E. coli strainMG1655 to obtain strain PS2419. The allele obtained by deletion of theileS gene, deltaileS203::kan, from strain IQ839 (Shiba and Schimmel,1992) is introduced in strain PS2419 by P1 transduction according to astandard protocol (Miller, 1972), and a transducant, strain PS 2449, isobtained in rich medium supplemented with kanamycin (25 mg/L) andarabinose (0.02%). In the same way, strain PS2306 is constructed byinsertion of the pVDC433 plasmid in strain MG1655 (resulting strain:PS27752) and P1 transduction of allele delta ileS203::kan in strainPS2752. Growth of the two strains deleted in the ileS locus, PS2306 andPS2449, requires the presence of arabinose, thus demonstrating thecontrolled expression of the wild type ileS gene (plasmid PVDC433,strain PS2306) and the mutated gene ileS alal 1 (plasmid pTLH33, strainPS2449) respectively.

Example 5 Sensitivity of the Isoleucyl-tRNA Synthetase to Non CanonicAmino Acids

[0132] Strains PS2306 and PS2449 are tested for their sensitivity toartificial amino acids whic present a steric resemblance to isoleucine.The cells are cultivated in MS mineral medium, succinate (0.2%),arabinose (0.2%) for 24 h at 37 degrees centigrade and diluted at{fraction (1/250)} in MS mineral medium. 0.5 ml of this cell suspensionare spread on a Petri dish containing 25 ml of MS medium supplementedwith succinate (0.2%) and arabinose (0.2%). A well is then prepared inthe middle of the dish and filled with 0.1 ml of an amino acid solution:

[0133] 1) 25 mM S-methyl cysteine

[0134] 2) 25 mM homocysteine

[0135] 3) 25 mM o-methyl-L-serine

[0136] 4) 25 mM Norleucine

[0137] 5) 25 mM Norvaline

[0138] The Petri dishes are then incubated at 37 degrees centigrade for24 hours and the appearance of a zone of inhibition around the well isrecorded. The diameters of the zones of attenuated growth are measuredas follows: TABLE 3 Amino acid PS2306 PS2449 S-methyl-L-cysteine 1, 3 cm3, 0 cm Homocysteine 2, 1 cm 2, 9 cm O-methyl-L-serine 2, 2 cm 5, 6 cmNorleucin 2, 9 cm 3, 4 cm Norvaline 3, 7 cm 8, 0 cm

[0139] All 5 non-canonic amino acids tested inhibit the growth of thetwo strains, but it is noted that in all cases there is a strongerinhibition on the strain expressing the mutated allele of the ileS genein the editing site. Thus the mutated isoleucyl-tRNA synthetase seems tohave an increased specificity for the substrate amino acid capable ofloading tRNAile with non-natural amino acids.

Example 6 Biochemical Characterization of the IleRS239-250Ala Mutant

[0140] The wild type and mutant IleRS enzymes were purified from strainsPS2306 and PS2449 by chromatography using the procedure described byHendrickson et al. (2002). Ile-tRNA loaced with the amino acid Val(Val-t-RNAIle) can be produced in large quantity by utilizing theediting mutant IleRS T242P (Hendrickson 2000). The utilzation of thisVal-tRNA/Ile has allowed testing of deacylation activity of severalenzymes using the protocol described by Hendrickson, 2000. The resultsobtained, shown in FIG. 4, clearly show that the IleRS239-250 mutant isdeficient in the deacylation function of Val-tRNAIle.

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[0157] 17. The 64 alleles of thyA with different codons at position 146of the coding sequence were constructed as follows. First, a unique NheIsite was introduced through a G429→A substitution in the thyA codingregion by site-directed mutagenesis (24) of plasmid pTSO (14) to yieldplasmid pTSO1. Oligonucleotides THY1(5′-CTGGATAAAATGGCGCTAGCACCGTGCCATGCATTC-3′) and THY2(5′-TCTGCCACATAGAACTGGAAGAATGCATGGCACGGT-3′) were used for this mutationwhich preserved the sense of the codon thus mutated. Plasmid pTSO1 wasthen digested with NheI and NsiI to remove from the thyA coding regionan 18 bp fragment containing codon 146 (UGC). All 64 oligonucleotides ofthe thyA coding sequence from nucleotides 427 to 444 and the 64oligonucleotides of the partial reverse sequence were constructed(GENAXIS Biotechnology, Montigny le Bretonneux, France). The 64 pairs ofcomplementary oligonucleotides were annealed and ligated with thedigested plasmid pTSO1.

[0158] 18. Cysteine gradient plates were done as described in FIG. 1Blegend. The effects of L-valine alone could not be directly examinedbecause exogenous L-valine is known to inhibit growth of E. coli K12 inminimal medium (25). This inhibition is relieved if L-isoleucine is alsosupplied. Thus, the Ile-Val dipeptide was used as a valine source,because this dipeptide is transported across the cell membrane and thenbroken down to isoleucine and valine (26).

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[0173] 33. mutS::spc+ allele (gift of F. Taddei, Hopital Necker-Enfants,Paris); ΔnrdD::kan+ allele, laboratory collection; AdnaQ::tet allele(gift of J. Shapiro, University of Chicago, Ill.); CU505(Δ(ilvE-ilvC)2049 leu455 galT1 IN(rrnD-rrnE)1) gift of Dr. M. Berlynfrom the E. coli Genetic Stock Center (New Haven, Conn.). Transfer ofgenes and of resistance markers by P1 transduction were carried outusing standard protocols (35).

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1 6 1 52 DNA Artificial Sequence synthetic oligonucleotide 1 ntagtaatcgcggcggcggc ggcggcggcg gcggcggcgg cgcgcgcaat at 52 2 50 DNA ArtificialSequence synthetic oligonucleotide 2 ngatattgcg cgcgccgccg ccgccgccgccgccgccgcc gccgcgatta 50 3 36 DNA Artificial Sequence syntheticoligonucleotide 3 ctggataaaa tggcgctagc accgtgccat gcattc 36 4 36 DNAArtificial Sequence synthetic oligonucleotide 4 tctgccacat agaactggaagaatgcatgg cacggt 36 5 33 DNA Artificial Sequence primer 5 ggggaattcggtgtgtgaaa ttgccgcaga acg 33 6 33 DNA Artificial Sequence primer 6ggcaagcttt caggtatttg ctgcccagat cga 33

We claim:
 1. A method to diversify the chemical composition of proteinsproduced in vivo by a cell comprising the step of disabling the editingfunction of at least one aminoacyl tRNA synthetase of the cell.
 2. Amethod for producing in vivo proteins comprising at least onenoncanonical amino acid comprising the step of: a) selecting a cellstrain wherein the editing function of at least one aminoacyl tRNAsynthetase of the cell has been disabled by mutagenesis, said disabledediting function allowing the aminoacyl tRNA synthetase to mischarge thecognate tRNA with said at least one noncanonical amino acid; b)culturing the selected strain in a culture medium comprising saidnoncanonical amino acid, or one of its precursors, under conditionsfavorable for the growth of said strain; and c) recovering from theculture medium or from the cells obtained in step b) the proteinscontaining said noncanonical amino acid.
 3. The method according toclaim 1 or 2, wherein the cell strain comprises a mutation in the DNAsequence encoding the editon domain of said disabled aminoacyl tRNAsynthetase compared to the wild type aminoacyl tRNA synthetase codingsequence.
 4. The method according to claim 3, wherein said DNA mutationleads to at least an amino acid mutation in the editing domain of saidaminoacyl tRNA synthetase.
 5. The method according to claim 3, whereinsaid DNA mutation leads to at least an amino acid substitution in theediting domain of said aminoacyl tRNA synthetase.
 6. The methodaccording to claim 5, wherein said mutation leads to a single amino acidsubstitution in the editing domain of said aminoacyl tRNA synthetase. 7.The method according to one of claims 1 or 2, wherein the disabledaminoacyl tRNA synthetase is capable of mischarging its cognate tRNAwith a canonical amino acid sterically similar to the amino acid chargedby the wild type aminoacyl tRNA synthetase.
 8. The method according toone of claims 1 or 2, wherein the disabled aminoacyl tRNA synthetase iscapable of mischarging its cognate tRNA with a noncanonical amino acidsterically similar to the amino acid charged by the wild type aminoacyltRNA synthetase on its cognate tRNA.
 9. A method for obtaining cellscapable of producing in vivo proteins comprising at least onenoncanonical amino acid comprising the step of mutagenizing the DNAsequence encoding the editing domain of an aminoacyl tRNA synthetase ina cell, said mutagenizing leading to an aminoacyl tRNA synthetasevariant having an amino acid mutation in its editing domain and saidmutation allowing the aminoacyl tRNA synthetase variant to mischarge itscognate tRNA with said at least one noncanonical amino acid.
 10. Amethod for obtaining cells capable of producing in vivo proteinscomprising at least one noncanonical amino acid according to claim 9,comprising the steps of: a) assaying the ability of an aminoacyl tRNAsynthetase variant having an amino acid mutation in its editing domainfor its ability to mischarge its cognate tRNA with a noncanonical aminoacid; b) mutagenizing the DNA sequence encoding the editing domain ofsaid aminoacyl tRNA synthetase in a cell, said mutagenizing leading tothe aminoacyl tRNA synthetase variant assayed in step a) and capable ofproducing detectable noncanonical amino acid mischarging; c) optionally,identifying, selecting and/or cloning the cells containing suchaminoacyl tRNA synthetase variant having the ability to mischarge onenoncanonical amino acid.
 11. The method of one of claims 1, 2, 9 or 10,wherein the cell is a microbial or animal cell.
 12. The method of claim11, wherein said microbial is a bacterium, a yeast or a fungus.
 13. Themethod of claim 12, wherein said bacterium is Escherichia coli orAcinetobacter.
 14. The method of one of claims 1, 2, 9 or 10, whereinthe editing domain of the aminoacyl tRNA synthetase has been disabled ormutagenized by homologous recombination.
 15. The method of one of claims1, 2, 9 or 10, wherein the editing domain of the aminoacyl tRNAsynthetase has been mutagenized by recombination into the genome of thetarget cell using an allelic replacement vector.
 16. A method forselecting an aminoacyl tRNA synthetase variant capable of mischargingits cognate tRNA with a noncanonical amino acid, comprising the stepsof: a) elaborating a DNA construct comprising a DNA sequence encoding anaminoacyl tRNA synthetase variant wherein said aminoacyl tRNA synthetasevariant has at least an amino acid mutation in its editing domaincompared with the wild type aminoacyl tRNA synthetase; b) transforming ahost cell with the DNA construct of step a) and, after a step ofculturing said transformed host cell, recovering and, optionally,purifying the recombinant aminoacyl tRNA synthetase variant expressed bythe host cell; c) assaying the ability of the recombinant aminoacyl tRNAsynthetase variant recovered in step b) for its ability to mischarge itscognate TRNA with a noncanonical amino acid; and d) selecting saidaminoacyl tRNA synthetase variant if detectable mischarging has beenproduced in step c).
 17. Isolated aminoacyl tRNA synthetase variantcapable of mischarging its cognate tRNA with a noncanonical amino acid,obtainable by the method of claim
 16. 18. Isolated nucleic acid sequenceencoded the aminoacyl tRNA synthetase variant of claim
 17. 19. Vectorcomprising a nucleic acid of claim
 18. 20. Transformed cell comprising anucleic acid encoding an aminoacyl tRNA synthetase variant of claim 17.21. Method for the production of proteins comprising a noncanonicalamino acid characterized in that said method comprises the followingsteps: a) culturing a cell of claim 20 in a culture medium comprisingthe noncanonical amino acid, or one of its precursors, capable of beingmischarged by the cognate tRNA of the aminoacyl tRNA synthetase variantcontained in said cell; and b) recovering the proteins comprising saidnoncanonical amino acid from the culture medium or from the cells ofstep a).
 22. An isolated protein comprising a noncanonical amino acidobtained by the method of claims 1, 2 or
 21. 23. The method of one ofclaims 1, 2, 9, 10 or 21, wherein the aminoacyl tRNA synthetase is anaminoacyl tRNA synthetase selected from the group consisting of theaminoacyl tRNA synthetase valyl-tRNA synthetase, isoleucyl-tRNAsynthetase, leucyl-tRNA synthetase, alanyl-tRNA synthetase, prolyl-tRNAsynthetase, threonyl-tRNA synthetase, phenyl-tRNA synthetase andlysyl-tRNA synthetase.